Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism

Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.     

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre. are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.     

Isolation and characterization of brassinosteroids from algal cultures of Chlorella vulgaris Beijerinck (Trebouxiophyceae)

The brassinosteroids (BRs) occur ubiquitously in the plant kingdom. The occurrence of BRs has been demonstrated in almost every part of higher plants, such as pollen, flower buds, fruits, seeds, vascular cambium, leaves, shoots and roots. In this study, BRs were isolated and identified in the culture of wild-type Chlorella vulgaris. Seven BRs, including teasterone, typhasterol, 6-deoxoteasterone, 6-deoxotyphasterol, 6-deoxocastasterone, castasterone and brassinolide, were identified by GC–MS. All compounds belong to the BR biosynthetic pathway. The results suggest that early and late C6 oxidation pathways are operating in C. vulgaris. This study represents the first isolation of BRs from C. vulgaris cultures.     

The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.     

Biosynthetic pathways of brassinolide in Arabidopsis

Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone --> 3-dehydro-6-deoxoteasterone --> 6-deoxotyphasterol --> 6-deoxocastasterone --> 6alpha-hydroxycastasterone --> castasterone --> BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone --> 3-dehydroteasterone --> typhasterol --> castasterone --> BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.     

Brassinosteroid levels increase drastically prior to morphogenesis of tracheary elements

As the first step toward understanding the involvement of endogenous brassinosteroids (BRs) in cytodifferentiation, we analyzed biosynthetic activities of BRs in zinnia (Zinnia elegans L. cv Canary Bird) cells differentiating into tracheary elements. The results of feeding experiments suggested that both the early and late C6-oxidation pathways occur during tracheary element differentiation. Gas chromatography-mass spectrometry analysis revealed that five BRs, castasterone, typhasterol, 6-deoxocastasterone, 6-deoxotyphasterol, and 6-deoxoteasterone, actually existed in cultured zinnia cells and culture medium. Quantification of endogenous BRs in each stage of tracheary element differentiation by gas chromatography-mass spectrometry exhibited that they increased dramatically prior to the morphogenesis, which was consistent with the idea that BRs are necessary for the initiation of the final stage of tracheary element differentiation. Moreover, the proportion of each BR in culture medium was quite different from that in cells, suggesting that specific BRs are selectively secreted into medium and may function outside the cells.     

Characterization of two brassinosteroid C-6 oxidase genes in pea

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.     

Brassinosteroid biosynthesis and inactivation

The term brassinosteroids (BRs) refers to the growth-promoting plant steroidal hormones. Various developmental programs including but not limited to cell elongation, stress tolerance, and skoto-/photo-morphogenesis are controlled by subnanomolar concentrations of BRs. Accordingly, BR mutants that are defective in BR biosynthetic or signaling pathways usually display dwarfism. Characterization of numerous BR dwarf mutants isolated from Arabidopsis , pea, tomato, and rice greatly contributed to our understanding of BR biology. Recently, an enzyme that mediates the final step in the BR biosynthetic pathways has been characterized by two different groups. The brassinolide synthases (Cytochrome P450s 85A2 and 85A3) are multifunctional enzymes that catalyze the last three consecutive steps in BR biosynthetic pathways, namely, C-6 hydroxylation, dehydrogenation, and Baeyer-Villiger type oxidation. In addition, many of the previously unknown steps have been genetically characterized. This review aims to summarize the knowledge that has been developed during the last 2–3 years in this field of BR biosynthesis and inactivation research.     

Brassinosteroids are inherently biosynthesized in the primary roots of maize, Zea mays L

GC-MS analysis revealed that primary roots of maize contain 6-deoxocathasterone, 6-deoxoteasterone and 6-deoxotyphasterol. These brassinosteroids, and the previously identified campesterol, campestanol, 6-deoxocastasterone and castasterone, in the roots are members of a biosynthetic pathway to castasterone, namely the late C-6 oxidation pathway, suggesting that its biosynthetic pathway is operative in the roots. To verify this, a cell-free enzyme extract was prepared from maize roots, and enzymatic conversions from campesterol to castasterone through the aforementioned sterols and brassinosteroids were examined. The presence for the biosynthetic sequences, campesterol-->24-methylcholest-4-en-3beta-ol-->24-methylcholest-4-en-3-one-->24-methylcholest-5 alpha-cholestan-3-one-->campestanol and 6-deoxoteasterone-->6-deoxo-3-dehydroteasterone-->6-deoxotyphasterol-->6-deoxocastasterone-->castasterone were demonstrated. These results indicate that maize roots contain a complete set of enzymes involved in the late C-6 oxidation pathway, thereby demonstrating that endogenous brassinosteroids are biosynthesized in the roots.     

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.     

C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5alpha-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5alpha-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.     

Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis

The conversion of castasterone (CS) to brassinolide (BL), a Baeyer-Villiger oxidation, represents the final and rate-limiting step in the biosynthesis of BL in plants. Heterologously expressed Arabidopsis thaliana CYP85A2 in yeast mediated the conversion of CS to BL as well as the C-6 oxidation of brassinosteroids (BRs). This indicated that CYP85A2 is a bifunctional enzyme that possesses BR C-6 oxidase and BL synthase activity. CYP85A2 is thus a cytochrome P450 that mediates Baeyer-Villiger oxidation in plants. Biochemical, physiological, and molecular genetic analyses of Arabidopsis CYP85A2 loss-of-function and overexpression lines demonstrated that CS has to be a bioactive BR that controls the overall growth and development of Arabidopsis plants. Mutant studies also revealed that BL may not always be necessary for normal growth and development but that Arabidopsis plants acquire great benefit in terms of growth and development in the presence of BL.     

shk1-D, a dwarf Arabidopsis mutant caused by activation of the CYP72C1 gene, has altered brassinosteroid levels

Brassinosteroids (BRs) are plant steroidal hormones that regulate plant growth and development. An Arabidopsis dwarf mutant, shrink1-D (shk1-D), was isolated and the phenotype was shown to be caused by activation of the CYP72C1 gene. CYP72C1 is a member of the cytochrome P450 monooxygenase gene family similar to BAS1/CYP734A1 that regulates BR inactivation. shk1-D has short hypocotyls in both light and dark, and short petioles and siliques. The seeds are also shortened along the longitudinal axis indicating CYP72C1 controls cell elongation. The expression of CPD, TCH4 and BAS1 were altered in CYP72C1 overexpression transgenic lines and endogenous levels of castasterone, 6-deoxocastasterone and 6-deoxotyphasterol were also altered. Unlike BAS1/CYP734A1 the expression of CYP72C1 was not changed by application of exogenous brassinolide. We propose that CYP72C1 controls BR homeostasis by modulating the concentration of BRs.     

Patterns of Dwarf expression and brassinosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development

Brassinosteroids (BRs) are essential for many physiological functions in plants, however little is known concerning where and when they are synthesized. This is especially true during flower and fruit production. To address this we have used a promoter-GUS reporter fusion and RT-PCR to determine the relative expression levels of the tomato Dwarf (D) gene that encodes a BR C-6 oxidase. In young seedlings GUS reporter activity was observed mainly in apical and root tissues undergoing expansion. In flowers GUS activity was observed in the pedicel joints and ovaries, whereas in fruits it was strongest during early seed development and was associated with the locular jelly and seeds. RT-PCR analysis showed that tissue-specific expression of Dwarf mRNA was consistent with that of the Dwarf:GUS fusion. In good correlation with the high local Dwarf activity, quantitative measurements of endogenous BRs indicated intense biosynthesis in developing tomato fruits, which were also found to contain high amounts of brassinolide. Grafting experiments showed the lack of BR transport indicating that BR action occurs at the site of synthesis.     

CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana

Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the ROTUNDIFOLIA3 (ROT3) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in Arabidopsis thaliana, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to ROT3, CYP90D1, and found that double mutants for ROT3 and CYP90D1 had a severe dwarf phenotype, whereas cyp90d1 single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. ROT3 and CYP90D1 were expressed differentially in leaves of A. thaliana, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of CYP90D1 was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.     

CYP90A1/CPD, a Brassinosteroid Biosynthetic Cytochrome P450 of Arabidopsis, Catalyzes C-3 Oxidation

Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.     

Biosynthesis and metabolism of brassinosteroids

Natural brassinosteroids so far identified from various plant species include biosynthetic congeners of brassinolide, such as cathasterone, teasterone, 3-dehydroteasterone, typhasterol and castasterone as well as another series of 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone, 6-deoxotyphasterol and 6-deoxocastasterone. Using cell culture system of Catharanthus roseus , the outlines of biosynthetic pathways of brassinolide, via plant sterol of campesterol, have now been demonstrated. There are two pathways, named early C6-oxidation pathway and late C6-oxidation pathway, both of which would be operating in wide varieties of plants. Metabolic studies with various plant systems revealed multiple paths of metabolism such as hydroxylation, epimerization, side chain cleavage, reduction and conjugation with glucose and fatty acids. Recent progress of biosynthesis and metabolism of brassinosteroids is described.     

Arabidopsis CYP85A2 catalyzes lactonization reactions in the biosynthesis of 2-deoxy-7-oxalactone brassinosteroids

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.     

Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea

Endogenous brassinosteroids (BRs) in the dwarf mutants lka and lkb of garden pea (Pisum sativum L.) and comparable wild-type plants were quantified by gas chromatography-selected ion monitoring using deuterated internal standards. In young shoots of the lkb mutant, the levels of brassinolide, castasterone, and 6-deoxocastasterone were 23-, 22-, and 9-fold lower, respectively, than those of wild-type plants. Applications of brassinolide, castasterone, typhasterol, 3-dehydroteasterone, and teasterone normalized internode growth of lkb seedlings. These findings indicate that the lkb plants are BR-deficient mutants, probably as a consequence of a block in the BR biosynthetic pathway prior to the production of teasterone. Young shoots of lka plants contained only 50% less brassinolide and 5 times more castasterone than the equivalent wild-type tissues. The lka seedlings were approximately 100 times less responsive to brassinolide than the lkb mutant, and application of castasterone had only a marginal effect on lka internode growth, suggesting that the lka lesion results in impaired sensitivity to BR.